PROJECT SUMMARY Mitochondria are dynamic organelles that undergo continuous fission and fusion. Mitochondrial dynamics are essential for cell survival, as well as for mitochondrial quality control, transport, distribution and inheritance. Defects in mitochondrial dynamics are implicated in various neurological disorders including Alzheimer?s, Parkinson?s and Huntington?s diseases, as well as in cardiovascular disease and cancer. The molecular mechanisms that accomplish mitochondrial membrane fission and fusion are poorly understood, as are the roles of the molecules involved in these processes. The long-term goal of this proposal is to address such issues. The mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1) is the master regulator of mitochondrial fission. Cytosolic Drp1 initiates mitochondrial fission via interactions with adaptor proteins, Mff, MiD49/51, or Fis1 localized at the mitochondrial surface. Subsequent Drp1 polymerization as ?helical scaffolds? around pre-destined mitochondrial division sites and GTP hydrolysis-driven scaffold constriction catalyzes mitochondrial fission. Exciting new studies have also necessitated a cooperative role for direct Drp1-phospholipid interactions, specifically with the mitochondrial lipid, cardiolipin (CL), in mitochondrial fission. However, very little is known about the cooperativity of Drp1-adaptor and Drp1-CL interactions, either in space or in time, during this process. Several unknown fundamental issues essential for understanding Drp1-mediated mitochondrial fission will be addressed in this application. These include 1) the mechanisms underlying Drp1 CL recognition, and the identity of Drp1 residues involved in specific phospholipid interactions, 2) the mechanism of the Drp1 variable domain (VD) in CL reorganization and nonbilayer phase transition, 3) the domain-specific topography of Drp1 on the membrane surface and conformational rearrangements that ensue upon specific adaptor and CL interactions, and 4) the cooperativity of CL and adaptor interactions in effecting mitochondrial fission. The proposed experiments will test the overarching hypothesis that cooperative Drp1 interactions with protein adaptors and CL promote the formation of a productive ?fission complex? that is localized in CL-rich micro-environments and drives membrane remodeling and fission through a Drp1 GTP hydrolysis-dependent CL bilayer-to-nonbilayer phase transition mechanism. We will use a tailor-made array of innovative fluorescence spectroscopic and microscopic approaches, coupled to solution and solid state NMR, to address these issues. These include the use of a novel variation of the FRET approach to determine domain-specific Drp1-membrane distances, collisional quenching of fluorescence to determine and measure Drp1 VD membrane insertion, and fluorescence imaging on model GUVs to visualize adaptor- and CL-regulated, Drp1-mediated membrane remodeling and fission. Successful outcomes of this research will provide (i) a fundamentally improved understanding of the cooperative molecular mechanisms underlying mitochondrial fission, and (ii) a molecular foundation for the design of drugs and therapeutics that can beneficially modulate mitochondrial dynamics under various disease states.